Watch tutorials, webinars and informative videos about PreSens optical sensor systems.
Oxygen and pH Effects of Modulator Agents on SK-MEL Cell Cultures
PhoxyCube enables multi-well dual analyte detection
Sandra Friedrich1, Barbara Goricnik1, Robert Johannes Meier2, Joachim Wegener1
1 Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany
2 PreSens Precision Sensing GmbH, Regensburg, Germany
Monitoring oxygen and pH levels is critical in cell culture, particularly for SK-MEL cells, a human melanoma cell line, used in this study. Oxygen is essential for cellular respiration and energy production, while pH affects enzyme activity, cellular metabolism, and overall viability. Deviations in O2 or pH can lead to metabolic stress, altered cell behavior, and inaccurate experimental results. In SK-MEL 28 cells, which are sensitive to microenvironment changes, maintaining optimal conditions is crucial for consistent growth, viability, and the accurate study of cancer-related processes such as metastasis and drug response.
This application note describes the simultaneous online detection of oxygen and pH in adherent SK-Mel cell culture by using the PreSens PhoxyCube. The PhoxyCube is a variable multi-well sensor reader that can measure dissolved oxygen and/or pH in multiple wells of different well plate formats during culture in the incubator. The optical O2 and pH sensors are read out in a non-invasive manner through the bottom of the plates. The measurement allows for visualization of cell proliferation kinetics and study the effects of metabolism manipulation of cell culture in real-time.
Materials & Methods
O2 and pH changes during culture were recorded using the PhoxyCube device (PreSens, Regensburg, Germany) with a 24-well plate with O2 and pH sensors in each individual well (PhoxyPlate 24 OP, see Fig. 1 B). The PhoxyPlates were pre-soaked and pre-equillibrated in 95 % air-saturated medium (measured pH of 7.6) at 37 °C in the incubator (5% CO2) for min. 3 hours. After the pre-equilibration phase a one-point adjustment for O2 (at 95 % a.s.) and for pH (at pH 7.6) was conducted with the PreSens PhoxyCube Reader Software. SK-MEL 28 cells were inoculated in each well of plate-columns 2 to 6 in Phenol red free DMEM growth medium (Gibco) + Glucose (500 µL with 4.5 g/L glucose) with a seeding density of 300 T/cm². Column 1 of the 24-well plate was kept with just growth medium without cells as blank control. After 24 hours the medium in all wells was exchanged with fresh growth medium.
48 hours after initial cell seeding, the growth medium was exchanged with Leibovitz´s L15-Medium without Phenol red (Gibco, short L15) L15-Glucose (blank without cells) in column 1, L15-glucose in column 2, L15- no glucose in column 3, L15-Glucose with Antimycin A as respiration blocking agent column 4, L15-Glucose with Malonobene as uncoupling agent in column 5, and L15-Glucose with a Pesticide (1 mM Acetamiprid, 4.45 %(v/v)) as unknown test substance in column 6. The cell adhesion and growth phase was recorded with a measurement interval of 20 min, the experimental phase (starting with media/substance exchange after 48 hours) was recorded with an interval of 5 min.
Results
During the attachment and growth phase all cell containing samples behave equally in both O2 consumption and pH change (see Fig. 2). The blank sample stays constant at pH 7.6 and 95 % a.s. After conducted cell attachment and growth for 48 hours, the actual experiment takes place. All test columns (with n = 4 experiments each) start at around 70 % a.s. and pH 6.6 after media and test substance addition. The blank experiment without cells results in a merely constant pH of 7.0 and constant pO2 around 100% air saturation (red graphs). The air saturation is higher than in the previous attachment phase, since the CO2 (5% v/v) in the incubator is switched off. SK-MEL cells in regular L15-Medium with Glucose addition (green graphs) show a slight and steady pH drop over 12 hours. The corresponding O2 curve shows a drop to 50 % a.s. after 1 hour and then slowly starts rising due to oxygen ingress. Cells in glucose-free L15-Medium depict a relatively constant pH level at pH 6.6, whereas the pO2 also drops in the first hour to a low of 40 % a.s. and then plateaus at 47 % a.s. after 2 hours (purple graphs).
The Antimycin A added to the cell culture (light blue graphs) results in a quick rise of O2 levels to air saturation within 2 hours due to the inhibition of the mitochondrial electron transport chain, specifically at complex III, leading to a sharp decrease in oxygen consumption as cellular respiration is blocked. This disruption also causes a drop in pH to pH 5.5 over the first 4 hours, as cells shift towards anaerobic glycolysis, producing lactic acid as a byproduct. Malonoben inhibits mitochondrial complex II, reducing oxygen consumption by impairing electron flow through the respiratory chain. This leads to a decrease in pH as cells compensate with increased anaerobic glycolysis, resulting in elevated lactate production. Therefore, the O2 level quickly drops to 22 % a.s. within the first hours and pH steadily drops to reach a plateau at pH 5.5 after approx. 9 hours (orange graphs). The pesticide Acetamiprid as test substance resulted in a quick rise in O2 levels to merely steady 90 % a.s. and a pH drop to pH 6.1 in the first two hours after start, where it stays constant (dark blue graphs).
Conclusion & Future Applications
As shown in this proof-of-concept report, the PhoxyCube monitoring platform allows detailed insight in every phase of cell culture experiments. Monitoring oxygen and pH simultaneously in real time gives the opportunity to observe metabolic changes imediately and, if required take according ajustments. In the experiments described here, PhoxyCube already demonstrated its value in monitoring cell culture and conducting toxicity / drug testing, but it can also contribute in other research areas. PhoxyCube is about to be used in hypoxia studies, tissue engineering and will speed up assay optimization (cell line, medium composition, etc.).